induction of systemic resistance in different varieties of...

Indian Journal of Experimental Biology

Vol 49 February 2011 pp 151-162

Induction of systemic resistance in different varieties of Solanum tuberosum by

pure and crude elicitor treatment

Himanshu S Bariya Vasudev R Thakkar Amit N Thakkar amp R B Subramanian

B amp R Doshi School of Biosciences Sardar Patel Maidan Sardar Patel University

Vadtal Road Bakrol Vallabh Vidyanagar 388 120 India

Received 28 January 2010 revised 13 July 2010

A 10 kD elicitor protein (infestin) produced by Phytopthora infestans was purified and its efficacy for induction of

systemic resistance in resistant and susceptible varieties of Solanum tuberosum was studied Culture filtrates from

P infestans with and without purified elicitor (infestin) were used as elicitors to understand the effect of purified elicitor

(infestin) on development of systemic resistance Culture filtrate and purified elicitor (infestin) were found to induce

hypersensitive reaction on the leaves of resistant varieties but not on susceptible varieties after 48 h Culture filtrate devoid

of purified elicitor (infestin) did not induce any necrotic spots even on resistant variety Purified elicitor (infestin) was found

to induce glucose oxidase NADPH oxidase superoxide dismutase glutathione reductase catalase and peroxidase enzymes

in resistant S tuberosum plants however the induction of these enzymes was low in susceptible varieties The oxidative

enzymes were found to induce earlier than antioxidative enzymes and there was negative correlation between these two

groups of enzymes Levels of salicylic acid phenylalanine ammonia lyase (PAL) β-1 3 glucanase and chitinase activities

were also found higher in resistant than in susceptible varieties It was observed that purified elicitor (infestin) was superior

to crude culture filtrate but was not capable of inducing systemic resistance in susceptible varieties

Keywords Defense related enzymes Elicitor Hypersensitivity response Phytophthora infestans Solanum tuberosum

In certain plant-pathogen interactions elicitors are

produced by pathogens Elicitors are highly specific

molecules produced by plant pathogens at low

concentration and are capable of inducing systemic

resistance in plants This requires standardization of

conditions for elicitation of systemic resistance in

crops According to gene-for-gene hypothesis

elicitors are products of avirulence (Avr) genes which

have complementary structure to products of

resistance (R) genes in plants In case of presence of

both of these dominant genes the plant-pathogen

interaction is termed as incompatible which results in

early recognition of pathogen by plant and induction

of HR followed by systemic acquired resistance

(SAR) which fights off the pathogen1 P infestans

produces such elicitor which has a potential to induce

systemic resistance in S tuberosum

Using hyphal wall components of P infestans2 and

also by treatment of lipoglycoprotein extracted from

it3 induction of systemic resistance has been

successfully generated in Solanum tuberosum plants

It was later found that arachidonic acid (AA) present

in lipoglycoprotein complex of hyphal wall is capable

of eliciting local but not systemic resistance in

S tuberosum plants45

Exogenous salicylic acid (SA)

also failed to generate effective resistance in potato

plants unlike Arabidopsis and tobacco6 Effective

elicitors for generating systemic resistance in potato

plants have been isolated from Phytopthora species

which are capable of inducing defense response at

nanomolar doses7 P infestans produces one of such

protein of 10 33 kD called purified elicitor (infestin)8 9

Aim of the present investigation was to study the

efficacy of purified elicitor (infestin) for recognition

of P infestans and instituting systemic resistance

against it in S tuberosum Efficacy of the purified

elicitor (infestin) was studied in comparison to crude

culture filtrate of P infestans

Materials and Methods

Plant material and fungusmdashFour different varieties

of potato (Solanum tuberosum L) plants obtained

from lsquoPotato Research Stationrsquo Deesa North Gujarat

India were grown in departmental backyard and

watered every 4 days Among these Badshah and

Bahar are the resistant varieties and Pukhraj and

________________

Correspondent author

Telephone +91-2692-234412 ext 304

Fax +91-2692-231041

E-mail vasuthakkargmailcom

INDIAN J EXP BIOL FEBRUARY 2011

152

Lavkar are the susceptible varieties P infestans was

isolated from infected potato tuber and maintained at

room temperature in the dark on V8 Juice agar plates

Chemicals and enzymesCytochrome C Xanthine

oxidase Xanthine NADPH salt etc were obtained

from Sisco Research Laboratory Pvt Ltd Mumbai

All other chemicals and solvents were obtained from

Qualigens Merck Rankem Himedia or Loba Chem

companies

Preparation of elicitorsThree types of elicitors

were used for treatments of plants Purified elicitor

(infestin) Fungal culture filtrate (FCF) and FCF

devoid of purified elicitor (infestin)

Purification of infestin from P infestansMycelial

agar plugs (7-8) were cut from 2-3 days old P

infestans grown on V8 Juice agar plates and

inoculated into liquid media (composition gl 30

glucose 7 peptone 1 yeast extract 001 thiamine

HCl 04 KHPO4 026 K2HPO4 01 MgSO47H2O 1

CaCl22H2O 1mg FeSO47H2O 0004 ZnSO47H2O

005 CuSO4 004 Na2MoO4 0045 MnCl2)

21 days old culture of P infestans was filtered

through three layers of muslin cloth and mixed with

one and half times volume of 100 acetone and

incubated at 4degC for 15 min Higher molecular weight

proteins were precipitated which were collected by

centrifugation of the solution at 10000 rpm for

10 min The supernatant was collected and mixed

with equal volume of 60 acetone and incubated at

4degC for 15 min The lower molecular weight proteins

were precipitated which were also obtained by

centrifugation at 10000 rpm for 10 min9 Presence of

purified elicitor (infestin) in the precipitated proteins

obtained after 60 acetone treatment was confirmed

by 10 SDS PAGE and a bioassay This 60 acetone

fraction containing purified elicitor (infestin) was

applied to Sephadex G 100 gel filtration column

(GFC) of 30 cm length and 1 cm width and ran with

potassium phosphate buffer (pH 72) at a flow rate of

1 mlmin Twelve fractions of 5 ml each were

collected and each fraction was ran in 12 SDS

PAGE along with low-range molecular marker

(Bangalore Genei) The fraction containing purified

elicitor (infestin) was identified and used as an elicitor

for giving treatment to plants

Fungal culture filtrate (FCF)Pinfestans was

grown in liquid medium as mentioned above under

static condition at 25degC in dark for 21 days and then

filtered using three layers of muslin cloth8 This

fungal culture filtrate (FCF) was used for treatment

FCF devoid of infestinFCF devoid of purified

elicitor (infestin) was prepared by collecting of all the

fractions of GFC except purified elicitor (infestin) and

mixed with higher molecular weight proteins [which

were obtained after treating culture filtrate with

100 acetone]

Treatment to plant About 100 microl of purified

elicitor (infestin) (098 mgml) fungal culture filtrate

(7505 mg proteinsml) and fungal culture filtrate

devoid of purified elicitor (infestin) (645 mg

proteinml) were applied to 40 days old four varieties

of potato (Badshah Bahar Pukhraj and Lavkar) on

6th leaf Plants were simultaneously infected by spores

of P infestans

Extraction of enzymesControl and treated leaves

(1g) were collected and homogenized with extraction

buffer (Potassium phosphate buffer pH 72 05 g PVP

and 005 mM PMSF) in pre-chilled pestle and mortar

The slurry was stirred mechanically for 5-10 min

Cellular debris was removed by centrifugation at

10000 rpm for 15 min Supernatant was collected and

used as crude enzyme Total proteins were determined

by Folin-Lowry estimation BSA served as blank

Enzyme assaysROS producing enzymes glucose

oxidase (GO)10

and NADPH oxidase11

were measured

spectrophotometrically Glutathione reductase12

superoxide dismutase13

catalase14

and peroxidase15

activities were measured using UV spectrophoto-

meter Phenylalanine ammonia lyase (PAL) activity

was measured by Hodgins method16

β-1 3 Glucanase

was measured using a method described by

Karthikeyan et al17

and chitinase enzyme activity was

measured by method of Monreal and Reese18

All

enzyme activities were expressed in terms of units

(U)g fresh wt (GFW) A correlation between

oxidative and antioxidative enzymes was found by

Studentrsquos t test19

Salicylic acid analysisLeaf tissues (05 g) were

collected at different time intervals after treatment

with three forms of elicitors The tissues were ground

in 5 ml of pre-chilled methanol using prechilled

mortar and pestle The slurry was collected in a vial

and kept overnight at 4degC to have complete extraction

of phenols It was then filtered through 02 micro

membrane at room temperature and concentrated by

keeping the vials open for 30 min Salicylic acid was

separated and estimated by high performance liquid

chromatography (HPLC) using Zorbex RP C18

column and mobile phase of acetate buffer (pH 55)

and methanol at a ratio of 7733 as described by

BARIYA et alINDUCTION OF SYSTEMIC RESISTANCE IN SOLANUM BY ELICITOR TREATMENT

153

Yalpani et al20

Pure salicylic acid at a concentration

of 1 mgml was used as a standard The detection was

carried out using UV detector at 315 nm

Results

Purification of infestin from PinfestansSDS -

PAGE separation of 60 acetone fraction from

21-day-old culture of P infestans showed presence of

an expected protein band of 10 kDa size For further

purification the acetone fraction was subjected to size

exclusion column chromatography The various

fractions collected were separated and visualized by

12 SDS-PAGE and the 10 kDa protein was found in

the 7th fraction (Fig 1) Protein estimation of various

elicitors showed 7505 mg proteinsml in fungal

culture filtrate 098 mg proteinml in purified elicitor

(infestin) and 645 mg proteinsml in fungal culture

filtrate devoid of infestin

BioassayAbout 100 microg of three types of

elicitors- i) purified elicitor (infestin) ii) FCF and iii)

culture filtrate devoid of purified elicitor (infestin)

were applied separately on the detached leaves of

different varieties of S tuberosum which showed

distinct reactions on resistant and susceptible

varieties Rapid macroscopic changes were observed

in purified elicitor (infestin) treated leaves which

resulted in chlorosis after 24 h and necrotic lesions

(HR) of about 02 mm diameter after 48 hours in

Badshah and Bahar (resistant) but not in leaves of

Pukhraj and Lavkar (susceptible) varieties (Fig 2 A)

Fig 2(A) HR reaction in resistant (BDBadshah and BH-Bahar) variety and absence of HR in susceptible variety (LKLavkar and

PKPukhraj) after treatment of purified purified elicitor (infestin) and (B) Conformation of R-Avr interaction - removal of purified

elicitor (infestin) from FCF results in absence of HR [1FCF treated leaf 2Purified elicitor (infestin) treated leaf 3Leaf treated

with FCF devoid of infestin and 4Control treated with potato dextrose broth]

Fig 1SDS-PAGE (12) stained with silver nitrate showing

separation of purified elicitin from P infestans [Lane 1FCF

without elicitin Lane 2fungal culture filtrate (FCF) Lane

3Purified elicitin and Lane 4Molecular marker with known

molecular weight


154

Application of FCF also showed similar HR only in

the leaves of resistant variety however the size and

number of necrotic lesions were less The R-gene

(andor its product) corresponding to avr gene product

(infestin) must have been present in the resistant

varieties which showed HR after treatment with P

infestans To confirm this we treated resistant leaves

with fungal culture filtrate devoid of purified elicitor

(infestin) and no necrotic lesion (HR) was found This

suggested that the purified elicitor (infestin) was

necessary for causing HR in resistant varieties which

was due to R-Avr interaction (Fig 2 B) These results

also showed that probably the purified elicitor

(infestin) would be capable of generating systemic

resistance only in resistant varieties not in susceptible

varieties To assess the ability of purified elicitor

(infestin) to elicit systemic resistance in S tuberosum

we measured activities of defense related enzymes

and concentration of salicylic acid

OxidativeROS generating enzymesActivities of

glucose oxidase (GO) and NADPH oxidase (Nox)

increased within 1 h of independent treatment of FCF

and purified elicitor (infestin) Increase in activities of

these enzymes was followed by gradual increase up to

21 days which signified massive HR (Fig 3) Level

of induction of these two ROS generating enzymes

was higher in resistant varieties compared to

susceptible varieties which was distinctly seen after

48 h (phenotypes of HR were also seen after 48 h in

bioassay) After 48 h of treatment with purified

elicitor (infestin) and culture filtrate GO was induced

in the range of 862plusmn003 to 108plusmn04 Unitg fresh wt

in resistant varieties from its basal activity of

167plusmn005 to 192plusmn003 Unitg fresh wt whereas in

susceptible varieties it was induced to 369plusmn02 to

446plusmn003 Unitg fresh wt from its basal activity of

09plusmn0021 to 092plusmn014 Unitg fresh wt Induction of

Nox in resistant varieties after 48 hrs was 85plusmn021 to


165plusmn023 to 176plusmn0013 Unitg fresh wt and in

susceptible varieties 293plusmn0051 to 333plusmn0019

Unitg fresh wt from its basal activity of 069plusmn027 to

076plusmn025 Unitg fresh wt after treatment with

purified elicitor (infestin) and culture filtrate

Induction of GO and Nox was low after treatment

with culture filtrate devoid of purified elicitor

(infestin) in all the varieties clearly indicating the role

of purified elicitor (infestin) in establishment of HR

AntioxidativeROS uptaking enzymesChanges in

activities of antioxidativeROS scavenging enzymes

[superoxide dismutase (SOD) glutathione reductase

(GR) catalase (CAT) and peroxidase (POX)] due to

treatment with various types of elicitors were

measured to study the kinetics of post HR Activities

of SOD GR CAT and POX were found to increase

compared to control in all four varieties after

treatment with purified elicitor (infestin) as well as

FCF Activities of these enzymes were initially lower

than oxidative enzymes However after HR

establishment (about 2-3 days after inocubation dpi)

the activities of these enzymes gradually increased

and remained elevated up to 21 days after infection

although activation was not that strong in susceptible

varieties (Figs 4 A and B) These results were

corroborated with lack of increase in SOD GR CAT

and POX activities in resistant varieties treated with

FCF devoid of purified elicitor (infestin)

Analysis of PR proteinsPhenylalanine ammonia

lyase (PAL) is one of the key regulators of SAR

because the conversion of phenylalanine to trans-

cinnamic acid serves as a branching point for

formation of lignin(s) and SA production via

phenylpropanoid pathways21

PAL activity increased

within 1h and remained elevated during the course of

infection in all four varieties (Fig 5) PAL activity

was found higher than all other defense enzymes in

control plants indicating PAL could be a major

constitutive enzyme that regulates SAR in potato

plant defense system PAL activity was not much

increased in plants treated with FCF devoid of

purified elicitor (infestin) indicating lack of signaling

in absence of Avr gene product β-13 Glucanase

(PR-2) and chitinase (PR-3) activities increased after

2 days of purified elicitor (infestin) or FCF treatment

in resistant varieties (Fig 6) Lack of increase of β-1

3 glucanase (PR-2) and chitinase (PR-3) activities in

susceptible varieties could be once again due to

absence of R-avr interaction No increase in these

enzymes activities were found after treatment with

FCF devoid of elicitor

Correlation between oxidative and antioxidative

enzymesmdashA significant and positive correlation was

observed between GO and Nox Significant but

negative correlation was observed between oxidative

and antioxidative enzyme activities GO and Nox

showed significant but negative correlation to

glutathione reductase and higher significance with

other antioxidative enzymes This analysis showed

that increase in activity of oxidative enzymes led to

decrease in activity of antioxidative enzyme and


155

Fig 3Activity of oxidative enzymes (glucose oxidase and NADPH oxidase) in all four varieties of potato at different time intervals

after treatment of different elicitors like purified elicitin (Eli) fungal culture filtrate (FCF) and fungal culture filtrate devoid of elicitin (F-

E) [Activity is expressed as Unitsg of fresh wt ABadshah BBahar CPukhraj and DLavkar]


156

Fig 4mdashActivity of antioxidative enzymes (glutathione reductase superoxide dismutase catalase and peroxidase) in (A) Badshah

(B) Bahar (C) Pukhraj and (D) Lavkar varieties of potato at different time intervals after treatment of different elicitors like purified

elicitin (Eli) fungal culture filtrate (FCF) and fungal culture filtrate devoid of elicitin (F-E) Activity is expressed as

Unitsg of fresh weight


157


158

vice-versa (Table 1) Activities of PAL chitinase and

glucanase also showed positive correlation with

antioxidative enzymes

Salicylic acid productionSA is thought to be

mobile signal for generation of systemic resistance in

plants Since PAL activity was found to increase after

treatment with purified elicitor (infestin) and FCF in

all four varieties SA was measured in all four

varieties of potato leaves treated with pure purified

elicitor (infestin) FCF and FCF devoid of purified

elicitor (infestin) after various time intervals SA

levels in control plants of all four varieties were same

However after treatment with purified elicitor

(infestin) and FCF accumulation of SA in resistant

varieties was higher compared to susceptible varieties

(Fig 7) In Badshah and Bahar (resistant) varieties

SA level increased to 27 to 40 mgg ( plusmn 0114) of

fresh tissue from 165 mgg ( plusmn 00062) in control

plants whereas in susceptible varieties it increased

only up to 63 mgg ( plusmn 00342)

Discussion

Response of different resistant and susceptible

varieties of S tuberosum plants to purified elicitor

(infestin) studied in the present work clarified

important concepts about the usage of elicitor to

induce systemic resistance in these plants It

demonstrated that purified elicitor (infestin) a small

peptide of 1033 kDa produced by P infestans after

21 days in culture was capable of inducing resistance

in resistant varieties of S tuberosum but not in

susceptible varieties P infestans or its hyphal wall

component can generate HR in potato tomato as well

as in tobacco has been reported21-23

We compared the

efficacy of the crude culture filtrate of P infestans

and an elicitor [avr gene product - purified elicitor

(infestin)] purified from it and tried to find

biochemical basis of their working Lack of HR in

susceptible varieties by treatment with FCF or

purified elicitor (infestin) could be due to lack of

specific R gene (andor its product) corresponding to

avr gene product (Infestin)

Ballvora et al1 have demonstrated the absence of R

gene in those susceptible varieties of S tuberosum

which are not showing necrotic lesions upon

treatment with P infestans Presence of R gene is

necessary for causing HR in S tuberosum has also

been shown by transformation of R1 gene in

susceptible variety which resulted in HR when treated

with P infestans1 Necrotic lesions observed in

bioassay were confirmed by studying kinetics of HR-

related enzymes Occurrence of HR was faster in

purified elicitor (infestin) compared to culture filtrate

Leaves of resistant varieties did not show HR

when treated with culture filtrate devoid of purified

elicitor (infestin) and eventually diseased state This

Fig 5mdashActivity of Phenylalanine ammonia Lyase (PAL) in all

four varieties of potato at different time intervals after treatment

of different elicitors like purified elicitin (Eli) fungal culture

filtrate (FCF) and fungal culture filtrate devoid of elicitin (F-E)

Activity is expressed as Unitsg of fresh weight


159

Fig 6mdashActivity of PR Proteins (β-1 3 Glucanase and Chitinase) in all four varieties of potato at different time intervals after treatment of

different elicitors like purified elicitin (Eli) fungal culture filtrate (FCF) and fungal culture filtrate devoid of elicitin (F-E) Activity is

expressed as Unitsg of fresh weight


160

suggested that purified elicitor (infestin) was

necessary for causing HR and development of SAR in

resistant varieties R and avr gene products interact

directly as receptor and ligand to activate plant

defense pathway22

Gene silencing of INF-1 (gene

responsible for producing elicitin) resulted in

deficiency of elicitin in P infestans and SAR has not

been observed when such strains are challenged to

potato and tomato plants23

Rapid induction of Nox and GO seen in purified

elicitor (infestin) and culture filtrate treated

S tuberosum confirmed that necrotic spots observed

in the detached leaf bioassay was HR Induction of

Nox resulted in HR by P infestans attack on

S tuberosum has also been observed by Yamamizo

et al24

Transgenic GO has been found to induce HR

in tobacco25

and potato plants26

Induction of these

oxidative enzymes due to treatment of elicitors was

higher in resistant (Badshah and Bahar) varieties

compared to susceptible (Pukhraj and Lavkar)

varieties The major role of GO in generating HR at

infection site could be due to its involvement in

pentose phosphate pathway through which plants

generate NADPH by oxidizing glucose27

Activity of

these two enzymes preceeded ROS scavenging

enzymes and showed rapid increase in the initial 48 h

after treatment of elicitors The hyphal wall

components of P infestans has also been known to

cause oxidative burst by generating ROS and thereby

inducing resistance in potato plants28

AntioxidativeROS scavenging enzymes are

activated by plants to stop the spreading HR lesions27

Increased activities of these enzymes were observed

in all four varieties following treatment with purified

elicitor (infestin) culture filtrate and culture filtrate

devoid of purified elicitor (infestin) Increased

activities of SOD GR CAT and POX in post-HR

status have also been reported elsewhere30

SOD is

responsible for generation of less toxic hydrogen

peroxide via dismutation of ROS and this hydrogen

peroxide is detoxified either by catalase or by

ascorbate-glutathione cycle followed by production of

water molecule28

Rate of increase in these enzymesrsquo

activity was higher in purified elicitor (infestin)

treated plants than in culture filtrate and least in case

of culture filtrate devoid of purified elicitor (infestin)

Moreover the rate of increase in the activities of these

enzymes became rapid after 2 days of treatment

showing their role in post-HR reactions The

difference between treatments of various elicitors was

visible till 28 days of application indicating better

capacity of purified elicitor (infestin) to induce

resistance than other elicitors in all varieties

Induction of all these enzymes was much lower in

susceptible varieties (Pukhraj and Lavkar) than in

resistant varieties (Badshah and Bahar) showing

differential induction of SAR in resistant and

susceptible varieties

Rapid increase in PAL activity was found in all

plants treated with different elicitors which was

maintained up to 28 days Accumulation of PAL

mRNA has been reported to occur within 30 min of

elicitor application29

Purified elicitor (infestin) was

found better inducer of PAL than other two elicitors

Increase in PAL was almost double in resistant

varieties than in susceptible verieties at initial hours

indicating significance of R-Avr interactions for its

activation However its rapid induction even before

Table 1Correlation between defense related enzymes activity after treatment of elicitin (purified elicitor) to potato plants

Glu-OX NOX GR SOD CAT POX PAL CHI GLU

Glu-OX 1

NOX 0126 1

GR -0113 -0097 1

SOD -0106 -0108 0041 1

CAT -0106 -0108 0045 0023 1

POX -0102 -0107 004 00137 0044 1

PAL -0129 -0163 0047 0027 0038 0059 1

CHI -0145 -0129 011 00251 0046 00281 0048 1

GLU -0105 -0105 0032 00138 0056 0054 003 0042 1

Significance at P=01

Numbers in dark background indicates a negative but significance correlation between two oxidative enzymes (Glucose oxidase and

NADPH Oxidase) and antioxidative enzymes (Glutathione reductase Superoxide Dismutase Catalase and Peroxidase) as well as

Phenylalanine ammonia Lyase chitinase and Glucanase

Glu-OX Glucose oxidase NOX NADPH oxidase GR Glutathione reductase CAT Catalase POX Peroxidase PAL

Phenylalanine ammonia Lyase CHI Chitinase and Glu β-1 3 Glucanase


161

HR establishment is another important question β-1

3 Glucanase and chitinase activities were found to

increase in resistant varieties after 2 days of treatment

of purified elicitor (infestin) and culture filtrate It has

been reported that in potato glucanase (PR-2) and

chitinase (PR-3) are two major PR proteins generated

during SAR30

Induction of these PR-proteins in the

present study was almost double in resistant varieties

than in susceptible varieties The present study

showed a significant correlation between oxidative

and antioxidative enzymes (Table 1)

Accumulation of SA in plants after treatment with

elicitor is one of the most important parameters

evaluated to determine induced systemic resistance31

In our study it was observed the induction of SA was

more in resistant varieties corroborating results of

PAL activity Increased SA level in resistant varieties

was capable of instituting SAR and its concentration

was in accordance with those reported by other

workers3233

Finally from above research findings it was

concluded that the treatment of potato plants with

purified elicitor (infestin) resulted in the development

of systemic resistance in resistant varieties but not in

susceptible varieties FCF devoid of purified elicitor

(infestin) did not cause HR lesions in resistant

varieties but resulted in disease Purified elicitor

(infestin) was found to induce SAR with more

intensity than crude FCF This work suggested that

purified elicitor (infestin) was effective for the

induction of HR and defense response in potato

plants but required potentiation to induce resistance

in susceptible plants

Acknowledgement We would like to dedicate this paper to Professor

I L Kothari who encouraged to undertake present

work We are thankful to Dr N H Patel Associate

Research Scientist Potato Research Station

Sardarkrushinagar Dantiwada Agricultural University

Deesa Dist Banaskantha India for providing four

different varieties of potato (Badshah Bahar Lavkar

and Pukhraj) for research purpose

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Fig 7mdashMeasurement of salicylic acid (SA) in all four varieties of

potato at different time intervals after treatment of different

elicitors like purified elicitin (Eli) fungal culture filtrate (FCF)

and fungal culture filtrate devoid of elicitin (F-E) SA level is

expressed as mgg of fresh weight of tissue


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biology edited by William V Dashek (CRC Press Boca

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457

11 Reusch V M amp Burger M M Distribution of marker enzymes

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249 (1974) 5337

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Chem 121 (1937) 561

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amp Samiyappan R Velazhahan R Induction of phenolics and

defence related enzymes in coconut (Cocos nucifera L) roots

treated with biocontrol agent Braz J Plant Physiol 18(3)

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Cell 3 (1991) 809

21 Nakane E Kawakita K Doke N amp Yoshioka H Elicitation

of primary and secondary metabolism during defense in the

potato J Gen Plant Pathol 69 (2003) 378

22 Park H J Doke N Miura Y Kawakita K Noritake T amp

Komatsubara H Induction of sub-systemic oxidative burst by

elicitor- stimulated local oxidative burst in potato plant

tissue A possible systemic signaling acquired resistance

Plant Sci 138 (1998) 197





24 Yamamizo C Kuchimura K Kobayashi A Katou S

Kawakita K Jones D G Doke N amp Yoshioka H Rewiring

mitogen-activated protein kinase cascade by positive

feedback confers potato blight resistance Plant Physiol 140

(2) (2006) 681

25 Kazan K Murray F Goulter K Llewellyn D amp Manners J

M Induction of cell death in transgenic plants expressing a

fungal glucose oxidase Mol plant Micr Inter 11 (1998) 555

26 Wu G Shortt B J Lawrence E B Levine E B Fitzsimmons

K C amp Shah D M Disease resistance confirmed by

expression of a gene encoding H2O2-generating glucose

oxidase in transgenic potato plant Plant Cell 8 (1995) 1357

27 Lu S Su W Li H amp Guo Z Abscisic acid improves drought

tolerance of triploid bermudagrass and involves H2O2 and

NO-induced antioxidant enzyme activities Plant Physiol

Biochem 47 (2009) 132

28 Fodor J Gullner G Adam A L Barna B Komives T amp

Kiraly Z Local and systemic responses of antioxidants to

tobacco mosaic virus infection and to salicylic acid in

tobacco Plant Physiol 114 (1997) 1443

29 Joos H J amp Hahlbrock K Phenylalanine ammonia-lyase in

potato (Solanum tuberosum L) Eur J Biochem 204 (1992)

621

30 Kombrink E Schroder M amp Hahlbrock K Several

ldquopathogenesis-relatedrdquo proteins in potato are 13-β-glucanase

and chitinase Proc Natl Acad Sci USA 85 (1988) 782



547

32 Raskin I Skubatz H Tang W amp Meeuse B J D Salicylic acid

levels in thermogenic and non-thermogenic plants Ann Bot

66 (1990) 373

33 Thakkar V R Subramanian R B amp Kothari I L Culture

filtrate of Lasiodiplodia theobromae restricts the

development of natural resistance in Brassica nigra plants

Indian J Exp Biol 42 (2004) 111


152

Lavkar are the susceptible varieties P infestans was

isolated from infected potato tuber and maintained at

room temperature in the dark on V8 Juice agar plates

Chemicals and enzymesCytochrome C Xanthine

oxidase Xanthine NADPH salt etc were obtained

from Sisco Research Laboratory Pvt Ltd Mumbai

All other chemicals and solvents were obtained from

Qualigens Merck Rankem Himedia or Loba Chem

companies

Preparation of elicitorsThree types of elicitors

were used for treatments of plants Purified elicitor

(infestin) Fungal culture filtrate (FCF) and FCF

devoid of purified elicitor (infestin)

Purification of infestin from P infestansMycelial

agar plugs (7-8) were cut from 2-3 days old P

infestans grown on V8 Juice agar plates and

inoculated into liquid media (composition gl 30

glucose 7 peptone 1 yeast extract 001 thiamine

HCl 04 KHPO4 026 K2HPO4 01 MgSO47H2O 1

CaCl22H2O 1mg FeSO47H2O 0004 ZnSO47H2O

005 CuSO4 004 Na2MoO4 0045 MnCl2)

21 days old culture of P infestans was filtered

through three layers of muslin cloth and mixed with

one and half times volume of 100 acetone and

incubated at 4degC for 15 min Higher molecular weight

proteins were precipitated which were collected by

centrifugation of the solution at 10000 rpm for

10 min The supernatant was collected and mixed

with equal volume of 60 acetone and incubated at

4degC for 15 min The lower molecular weight proteins

were precipitated which were also obtained by

centrifugation at 10000 rpm for 10 min9 Presence of

purified elicitor (infestin) in the precipitated proteins

obtained after 60 acetone treatment was confirmed

by 10 SDS PAGE and a bioassay This 60 acetone

fraction containing purified elicitor (infestin) was

applied to Sephadex G 100 gel filtration column

(GFC) of 30 cm length and 1 cm width and ran with

potassium phosphate buffer (pH 72) at a flow rate of

1 mlmin Twelve fractions of 5 ml each were

collected and each fraction was ran in 12 SDS

PAGE along with low-range molecular marker

(Bangalore Genei) The fraction containing purified

elicitor (infestin) was identified and used as an elicitor

for giving treatment to plants

Fungal culture filtrate (FCF)Pinfestans was

grown in liquid medium as mentioned above under

static condition at 25degC in dark for 21 days and then

filtered using three layers of muslin cloth8 This

fungal culture filtrate (FCF) was used for treatment

FCF devoid of infestinFCF devoid of purified

elicitor (infestin) was prepared by collecting of all the

fractions of GFC except purified elicitor (infestin) and

mixed with higher molecular weight proteins [which

were obtained after treating culture filtrate with

100 acetone]

Treatment to plant About 100 microl of purified

elicitor (infestin) (098 mgml) fungal culture filtrate

(7505 mg proteinsml) and fungal culture filtrate

devoid of purified elicitor (infestin) (645 mg

proteinml) were applied to 40 days old four varieties

of potato (Badshah Bahar Pukhraj and Lavkar) on

6th leaf Plants were simultaneously infected by spores

of P infestans

Extraction of enzymesControl and treated leaves

(1g) were collected and homogenized with extraction

buffer (Potassium phosphate buffer pH 72 05 g PVP

and 005 mM PMSF) in pre-chilled pestle and mortar

The slurry was stirred mechanically for 5-10 min

Cellular debris was removed by centrifugation at

10000 rpm for 15 min Supernatant was collected and

used as crude enzyme Total proteins were determined

by Folin-Lowry estimation BSA served as blank

Enzyme assaysROS producing enzymes glucose

oxidase (GO)10

and NADPH oxidase11

were measured

spectrophotometrically Glutathione reductase12

superoxide dismutase13

catalase14

and peroxidase15

activities were measured using UV spectrophoto-

meter Phenylalanine ammonia lyase (PAL) activity

was measured by Hodgins method16

β-1 3 Glucanase

was measured using a method described by

Karthikeyan et al17

and chitinase enzyme activity was

measured by method of Monreal and Reese18

All

enzyme activities were expressed in terms of units

(U)g fresh wt (GFW) A correlation between

oxidative and antioxidative enzymes was found by

Studentrsquos t test19

Salicylic acid analysisLeaf tissues (05 g) were

collected at different time intervals after treatment

with three forms of elicitors The tissues were ground

in 5 ml of pre-chilled methanol using prechilled

mortar and pestle The slurry was collected in a vial

and kept overnight at 4degC to have complete extraction

of phenols It was then filtered through 02 micro

membrane at room temperature and concentrated by

keeping the vials open for 30 min Salicylic acid was

separated and estimated by high performance liquid

chromatography (HPLC) using Zorbex RP C18

column and mobile phase of acetate buffer (pH 55)

and methanol at a ratio of 7733 as described by


153

Yalpani et al20




Results


































molecular weight


154







































































































155





156






157


158





















Discussion













We compared the





























159





160





defense pathway22












et al24


in tobacco25

and potato plants26

Induction of these








Activity of















SOD is





water molecule28






























Glu-OX 1

NOX 0126 1

GR -0113 -0097 1

SOD -0106 -0108 0041 1

CAT -0106 -0108 0045 0023 1

POX -0102 -0107 004 00137 0044 1

PAL -0129 -0163 0047 0027 0038 0059 1

CHI -0145 -0129 011 00251 0046 00281 0048 1

GLU -0105 -0105 0032 00138 0056 0054 003 0042 1








161







during SAR30














workers3233






















References



















162






(1989) 651








547



Crop Sci 44 (2004) 1920











457



249 (1974) 5337








Chem 244 (1969) 6049


Chem 121 (1937) 561



New York) 1955 773


Chem 246 (1971) 2977





(2006) 367








Cell 3 (1991) 809








Plant Sci 138 (1998) 197









(2) (2006) 681











Biochem 47 (2009) 132







621






547



66 (1990) 373






153

Yalpani et al20




Results


































molecular weight


154







































































































155





156






157


158





















Discussion













We compared the





























159





160





defense pathway22












et al24


in tobacco25

and potato plants26

Induction of these








Activity of















SOD is





water molecule28






























Glu-OX 1

NOX 0126 1

GR -0113 -0097 1

SOD -0106 -0108 0041 1

CAT -0106 -0108 0045 0023 1

POX -0102 -0107 004 00137 0044 1

PAL -0129 -0163 0047 0027 0038 0059 1

CHI -0145 -0129 011 00251 0046 00281 0048 1

GLU -0105 -0105 0032 00138 0056 0054 003 0042 1








161







during SAR30














workers3233






















References



















162






(1989) 651








547



Crop Sci 44 (2004) 1920











457



249 (1974) 5337








Chem 244 (1969) 6049


Chem 121 (1937) 561



New York) 1955 773


Chem 246 (1971) 2977





(2006) 367








Cell 3 (1991) 809








Plant Sci 138 (1998) 197









(2) (2006) 681











Biochem 47 (2009) 132







621






547



66 (1990) 373






154







































































































155





156






157


158





















Discussion













We compared the





























159





160





defense pathway22












et al24


in tobacco25

and potato plants26

Induction of these








Activity of















SOD is





water molecule28






























Glu-OX 1

NOX 0126 1

GR -0113 -0097 1

SOD -0106 -0108 0041 1

CAT -0106 -0108 0045 0023 1

POX -0102 -0107 004 00137 0044 1

PAL -0129 -0163 0047 0027 0038 0059 1

CHI -0145 -0129 011 00251 0046 00281 0048 1

GLU -0105 -0105 0032 00138 0056 0054 003 0042 1








161







during SAR30














workers3233






















References



















162






(1989) 651








547



Crop Sci 44 (2004) 1920











457



249 (1974) 5337








Chem 244 (1969) 6049


Chem 121 (1937) 561



New York) 1955 773


Chem 246 (1971) 2977





(2006) 367








Cell 3 (1991) 809








Plant Sci 138 (1998) 197









(2) (2006) 681











Biochem 47 (2009) 132







621






547



66 (1990) 373






155





156






157


158





















Discussion













We compared the





























159





160





defense pathway22












et al24


in tobacco25

and potato plants26

Induction of these








Activity of















SOD is





water molecule28






























Glu-OX 1

NOX 0126 1

GR -0113 -0097 1

SOD -0106 -0108 0041 1

CAT -0106 -0108 0045 0023 1

POX -0102 -0107 004 00137 0044 1

PAL -0129 -0163 0047 0027 0038 0059 1

CHI -0145 -0129 011 00251 0046 00281 0048 1

GLU -0105 -0105 0032 00138 0056 0054 003 0042 1








161







during SAR30














workers3233






















References



















162






(1989) 651








547



Crop Sci 44 (2004) 1920











457



249 (1974) 5337








Chem 244 (1969) 6049


Chem 121 (1937) 561



New York) 1955 773


Chem 246 (1971) 2977





(2006) 367








Cell 3 (1991) 809








Plant Sci 138 (1998) 197









(2) (2006) 681











Biochem 47 (2009) 132







621






547



66 (1990) 373






156






157


158





















Discussion













We compared the





























159





160





defense pathway22












et al24


in tobacco25

and potato plants26

Induction of these








Activity of















SOD is





water molecule28






























Glu-OX 1

NOX 0126 1

GR -0113 -0097 1

SOD -0106 -0108 0041 1

CAT -0106 -0108 0045 0023 1

POX -0102 -0107 004 00137 0044 1

PAL -0129 -0163 0047 0027 0038 0059 1

CHI -0145 -0129 011 00251 0046 00281 0048 1

GLU -0105 -0105 0032 00138 0056 0054 003 0042 1








161







during SAR30














workers3233






















References



















162






(1989) 651








547



Crop Sci 44 (2004) 1920











457



249 (1974) 5337








Chem 244 (1969) 6049


Chem 121 (1937) 561



New York) 1955 773


Chem 246 (1971) 2977





(2006) 367








Cell 3 (1991) 809








Plant Sci 138 (1998) 197









(2) (2006) 681











Biochem 47 (2009) 132







621






547



66 (1990) 373






157


158





















Discussion













We compared the





























159





160





defense pathway22












et al24


in tobacco25

and potato plants26

Induction of these








Activity of















SOD is





water molecule28






























Glu-OX 1

NOX 0126 1

GR -0113 -0097 1

SOD -0106 -0108 0041 1

CAT -0106 -0108 0045 0023 1

POX -0102 -0107 004 00137 0044 1

PAL -0129 -0163 0047 0027 0038 0059 1

CHI -0145 -0129 011 00251 0046 00281 0048 1

GLU -0105 -0105 0032 00138 0056 0054 003 0042 1








161







during SAR30














workers3233






















References



















162






(1989) 651








547



Crop Sci 44 (2004) 1920











457



249 (1974) 5337








Chem 244 (1969) 6049


Chem 121 (1937) 561



New York) 1955 773


Chem 246 (1971) 2977





(2006) 367








Cell 3 (1991) 809








Plant Sci 138 (1998) 197









(2) (2006) 681











Biochem 47 (2009) 132







621






547



66 (1990) 373






158





















Discussion













We compared the





























159





160





defense pathway22












et al24


in tobacco25

and potato plants26

Induction of these








Activity of















SOD is





water molecule28






























Glu-OX 1

NOX 0126 1

GR -0113 -0097 1

SOD -0106 -0108 0041 1

CAT -0106 -0108 0045 0023 1

POX -0102 -0107 004 00137 0044 1

PAL -0129 -0163 0047 0027 0038 0059 1

CHI -0145 -0129 011 00251 0046 00281 0048 1

GLU -0105 -0105 0032 00138 0056 0054 003 0042 1








161







during SAR30














workers3233






















References



















162






(1989) 651








547



Crop Sci 44 (2004) 1920











457



249 (1974) 5337








Chem 244 (1969) 6049


Chem 121 (1937) 561



New York) 1955 773


Chem 246 (1971) 2977





(2006) 367








Cell 3 (1991) 809








Plant Sci 138 (1998) 197









(2) (2006) 681











Biochem 47 (2009) 132







621






547



66 (1990) 373






159





160





defense pathway22












et al24


in tobacco25

and potato plants26

Induction of these








Activity of















SOD is





water molecule28






























Glu-OX 1

NOX 0126 1

GR -0113 -0097 1

SOD -0106 -0108 0041 1

CAT -0106 -0108 0045 0023 1

POX -0102 -0107 004 00137 0044 1

PAL -0129 -0163 0047 0027 0038 0059 1

CHI -0145 -0129 011 00251 0046 00281 0048 1

GLU -0105 -0105 0032 00138 0056 0054 003 0042 1








161







during SAR30














workers3233






















References



















162






(1989) 651








547



Crop Sci 44 (2004) 1920











457



249 (1974) 5337








Chem 244 (1969) 6049


Chem 121 (1937) 561



New York) 1955 773


Chem 246 (1971) 2977





(2006) 367








Cell 3 (1991) 809








Plant Sci 138 (1998) 197









(2) (2006) 681











Biochem 47 (2009) 132







621






547



66 (1990) 373






160





defense pathway22












et al24


in tobacco25

and potato plants26

Induction of these








Activity of















SOD is





water molecule28






























Glu-OX 1

NOX 0126 1

GR -0113 -0097 1

SOD -0106 -0108 0041 1

CAT -0106 -0108 0045 0023 1

POX -0102 -0107 004 00137 0044 1

PAL -0129 -0163 0047 0027 0038 0059 1

CHI -0145 -0129 011 00251 0046 00281 0048 1

GLU -0105 -0105 0032 00138 0056 0054 003 0042 1








161







during SAR30














workers3233






















References



















162






(1989) 651








547



Crop Sci 44 (2004) 1920











457



249 (1974) 5337








Chem 244 (1969) 6049


Chem 121 (1937) 561



New York) 1955 773


Chem 246 (1971) 2977





(2006) 367








Cell 3 (1991) 809








Plant Sci 138 (1998) 197









(2) (2006) 681











Biochem 47 (2009) 132







621






547



66 (1990) 373






161







during SAR30














workers3233






















References



















162






(1989) 651








547



Crop Sci 44 (2004) 1920











457



249 (1974) 5337








Chem 244 (1969) 6049


Chem 121 (1937) 561



New York) 1955 773


Chem 246 (1971) 2977





(2006) 367








Cell 3 (1991) 809








Plant Sci 138 (1998) 197









(2) (2006) 681











Biochem 47 (2009) 132







621






547



66 (1990) 373






162






(1989) 651








547



Crop Sci 44 (2004) 1920











457



249 (1974) 5337








Chem 244 (1969) 6049


Chem 121 (1937) 561



New York) 1955 773


Chem 246 (1971) 2977





(2006) 367








Cell 3 (1991) 809








Plant Sci 138 (1998) 197









(2) (2006) 681











Biochem 47 (2009) 132







621






547



66 (1990) 373





induction of systemic resistance in different varieties of...

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